Teratogenic effect of alcohol has been documented over the years. It is known that maternal alcohol abuse during gestation can result in various fetal injuries, including intrauterine growth retardation, craniofacial dysmorphology, and central nervous system damage. While the developmental defects from alcohol abuse during gestation have been described, it is still unanswered about what are the specific mechanisms by which alcohol mediates these injuries. This is important question to address in order for us to identify affected children at an early age and intervene to prevent or mitigate the damage. It has been reported that alcohol consumption alters fetal DNA methylation in mice. However, due to the inherent limitations of studies conducted in humans, the use of alternative models is important in addressing these unanswered questions. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. Central hypotheses being tested in this application are (1) alcohol induces epigenetic changes in human embryonic stem cells that significantly affect gene regulatory networks in stem cell self-renewal/differentiation and (2) molecular regulation in multipotent adult dental pulp stem cells is significantly altered by alcohol- induced epigenetic changes. The immediate objective of this application is to define molecular signatures and pathways that are affected by alcohol in human embryonic and adult dental pulp stem cells. As an exploratory study, we propose a three-year grant to perform systems biology analysis with combined DNA methylomic and transcriptomic profiling of molecular signatures and pathways affected by alcohol. The importance of the orchestrated interplay between molecular regulators has been demonstrated in the maintenance of self-renewing pluripotent property or the initiation of differentiation in stem cells. Epigenetic factors play important roles in controlling these molecular regulators. Specific Aims proposed in this application will provide us with a novel opportunity that has never been explored. Outcomes from these aims will significantly advance our understanding in human stem cell biology by elucidating epigenetic molecular signatures and pathways affected by alcohol exposure in human embryonic stem cells and human dental pulp stem cells. Stem cells offer powerful tools to elucidate mechanisms underlying normal biological functions and the mechanisms by which alcohol disrupts normal functions in susceptible organs. Our understanding on the effect of alcohol on stem cells (especially adult stem cells proposed for regenerative medicine) will be beneficial to design future clinical application of stem cells to improve human health. PUBLIC HEALTH RELEVANCE: Alcohol abuse is known to cause abnormalities in a number of organ systems. Disorders most commonly associated with chronic alcohol consumption include alcoholic liver disease (ALD), pancreatitis, cardiovascular disease, endocrine dysfunction, osteoporosis, cancer, and immune dysfunction. It is still unanswered about what are the specific mechanisms by which alcohol induces developmental defects. This is important question to address in order for us to identify affected children at an early age and intervene to prevent or mitigate the damage. Outcomes from this proposal will significantly advance our understanding in human stem cell biology by elucidating epigenetic molecular signatures and pathways affected by alcohol exposure in human embryonic stem cells and human dental pulp stem cells. Stem cells may also prove valuable in developing therapeutic approaches for treating alcohol-induced physiological impairments and overt organ damage. Our understanding on the effect of alcohol on stem cells will be beneficial to design future clinical application of stem cells to improve human health.